Antibiotic 67-121, a polyene antifungal antibiotic produced by actinoplanes caeruleus

ABSTRACT

A microorganism of the genus Actinoplanes produces a novel complex of polyene antibiotics herein designated collectively Antibiotic 67-121 (and sometimes referred to as the Antibiotic 67-121 complex). The individual components are designated Antibiotic 67-121A, Antibiotic 67-121B, Antibiotic 67-121C and Antibiotic 67-121D. The individual components have ultraviolet spectra characteristic of heptaenes. Further, they are orally active against a variety of strains of Candida albicans including many clinical isolates thereof.

This application is a continuation-in-part of copending application Ser. No. 450,040, filed Mar. 11, 1974 now abandoned.

This invention relates to novel antibiotically active substances, to their preparation and to pharmaceutical compositions containing such substances. More specifically this invention relates to a novel polyene antibiotic complex and the component antibiotics thereof obtainable by cultivation of a hitherto undescribed microorganism of the genus Actinoplanes of the family Actinoplanceae.

A general discussion of macrolide antibiotics is to be found in Kirk-Othmer, Encyclopedia of Chemical Technology, 2nd Edition, Volume 12, pp. 632 et seq. As specified therein, antibiotics having the common feature of a macrocyclic lactone ring are commonly classified as a distinct group of antibiotics, generally termed the macrolide antibiotics. In addition to the lactone ring, the macrolide group of antibiotics are further distinguished by having various ketonic and hydroxyl functions and glycosidically bound deoxy sugars. One sub-grouping is that designated the antibacterial antibiotics. A further sub-grouping, typified by antifungal activity, is that designated the polyene macrolide antibiotics, or more briefly the polyene antibiotics, this sub-grouping being distinguished by having a conjugated double system of from 3 to 7 conjugated bonds. Identification of an antibiotic as a polyene antibiotic is facilitated by the characteristic ultraviolet absorption spectrum. As representative polyene antibiotics there may be mentioned amphotericin A, amphotericin B, candicidin, nystatin, perimycin, pimaricin and trichomycin. A general description of polyene antibiotics is to be found in Kirk-Othmer, Encyclopedia of Chemical Technology, 2nd Edition, Volume 16, pp. 133 et seq., while data on the ultraviolet absorption maxima for the above named, and other, polyene antibiotics is given in Umezawa, H. et al., Index of Antibiotics from Actinomycetes, University of Tokyo Press, 1967, pp. 879- 885.

In one of its aspects, the present invention provides a novel polyene antibiotic complex designated the antibiotic 67-121 complex, and the component antibiotics thereof herein designated antibiotic 67-121A, 67-121B, 67-121C and 67-121D. These antibiotics have been found to exhibit useful antifungal activity and in specific tests against a variety of strains of Candida albicans to possess enhanced antifungal activity as compared with the known polyene antibiotic candicidin. The novel antibiotic 67-121 complex and its component antibiotics are obtainable by cultivation of a hitherto undescribed micro-organism of the genus Actinoplanes.

In another of its aspects, the present invention provides a process for the preparation of an antibiotic complex designated the antibiotic 67-121 complex, or an antibiotic component thereof designated 67-121A, 67-121B, 67-121C. and 67-121D, which process comprises cultivating an Antibiotic 67-121 complex - or component - producing microorganism of the genus Actinoplanes in a nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to the medium. Specifically, we have employed a microorganism of a new species designated herein Actinoplanes caeruleus.

The Micro-Organism

The specific microorganism employed herein was isolated from a soil sample from a sage brush area in Bristol Cone Park, California, and was identified as a member of the family Actinoplanaceae, genus Actinoplanes, and more specifically as a member of a new species designated Actinoplanes caeruleus. A representative culture of the microorganism designated Actinoplanes caeruleus has been deposited and is now a part of the permanent collection of the Northern Utilization Research and Development Division, United States Department of Agriculture, Peoria, Illinois, U.S.A., where it has been assigned accession number NRRL 5325. Sub-cultures of Actinoplanes caeruleus NRRL 5325 are available upon request to the aforementioned agency. In addition to the strain of Actinoplanes caeruleus deposited, this invention embraces mutants and variants thereof that exhibit the same or substantially the same taxonomical and physiological properties of the deposited strain which properties include the production of Antibiotic 67-121 complex.

The microorganism employed herein was classifiable as being of the genus Actinoplanes on the basis of its producing no aerial mycelium and forming globose to digitate sporangia which upon dehiscence release globuse motile spores having a tuft of polar flagella. The micro-organism exhibited characteristics sufficiently different from known species of Actinoplanes, as hereinafter shown, to warrant designation as a new species. The species was designated Actinoplanes caeruleus on the basis of the formation of deep blue to blue-green colonies when grown on a variety of media. A surprising, advantageous and distinguishing feature of Actinoplanes caeruleus is the ability to produce the novel polyene antibiotic complex, and component antibiotics, herein designated 67-121.

Actinoplanes caeruleus is aerobic and grows well on a variety of nutrient media at from 26° to 37° C. At 45° C. and above substantially no growth is observed. Preferably, in the production of antibiotic, cultivation is effected under submerged aerobic conditions at a pH of from 6.5 to 8.0. Actinoplanes caeruleus is sensitive to sodium chloride, growing very poorly in media containing 1.5% sodium chloride. It reduces nitrate to nitrite, partially liquifies gelatin, very slowly decomposes cellulose and only slightly hydrolyses starch.

Other taxanomical data for Actinoplanes caeruleus are set forth in Tables I- VI as follows:

    ______________________________________                                         Table I  --    Colony characteristics on various                                              media                                                           Table II --    Carbohydrate utilisation                                        Table III                                                                               --    Nitrogen utilisaton                                             Table IV --    Comparison of growth characteristics                                           of A. caeruleus with other Actinoplanes -  species              Table V  --    Comparison of physiological character-                                         istics of A. caeruleus with other                                              Actinoplanes species                                            Table VI --    Comparison of carbohydrate utilisation                                         of A. caeruleus with other Actinoplanes                                        species.                                                        ______________________________________                                    

In Tables I and III the color descriptions given consist of two designations. The first color designation is a color name taken from the "Descriptive Color Name Dictionary", by Taylor, Knoche and Granville, published by the Container Corporation of America, 1950 (U.S.A.), in combination with a color chip number corresponding to the color name, the chip number being taken from the "Color Harmony Manual", 4th Edition, 1958, published by the Container Corporation of America, U.S.A. The second color designation is a synonym or near synonym of the first designation and is taken from the National Bureau of Standards, Circular 553, November 1955 (U.S.A.).

In Table II the medium employed consisted of 1% of the specified carbohydrate, 0.5% yeast extract, 1.5% agar and the remainder water, while in Table III the medium employed consisted of the stated amount of the nitrogen source, 1% glucose, 1.5% agar and the remainder water.

Information for Actinoplanes species, other than Actinoplanes caeruleus, given in comparative Tables IV to VI was obtained from the following sources:

(a) for A. philippinensis, A. brasiliensis, A. missouriensis, A. utahensis from Thieman J. E. et. al., Journal of Antibiotics 22 (1969) pp 119-125,

(b) for A. sp NRRL 3884 from DOS Pat. No. 2,252,937, and

(c) for A. italacus from Beretta G., International Journal Systematic Bacteriology 23 (1973) pp 37-42.

                  TABLE I                                                          ______________________________________                                         Colony Observations on Various Media                                           Medium           Observations                                                  ______________________________________                                         Glucose     Growth:  good, plicate, no aerial                                  Asparagine           mycelium, no diffusible                                   Agar                 pigment                                                               Color:   g41c dusty orange, moderate                                                    orange 53                                                 NZ Amine *  Growth:  good, plicate, no aerial                                  Glucose              mycelium, no diffusible                                   Agar                 pigment                                                               Color:   g5pa bright orange, vivid                                                      orange 48                                                 Milk        Growth:  good, no aerial mycelium,                                                      no diffusible pigment, no                                                      hydrolysis (or in some cul-                                                    tures hydrolysis only be-                                                      neath colonies)                                                       Color:   g4na bright orange, vivid                                                      orange 48 plicate                                         Bennet's    Growth:  good, plicate, no aerial                                  Agar                 mycelium, no diffusible                                                        pigment                                                               Color:   g3ne topaz, strong yellowish                                                   brown 74 and some darker mot-                                                  tled areas of a dark green-                                                    ish black                                                 Emerson's   Growth:  good, plicate, no aerial                                  Agar                 mycelium, no diffusible                                                        pigment                                                               Color:   g3ne topaz, strong yellowish                                                   brown 74                                                  Tomato Paste                                                                               Growth:  Fair to good, folded, no                                  Oatmeal Agar         aerial mycelium, no diffus-                                                    ible pigment                                                          Color:   mixture of g3ea light melon                                                    yellow, pale orange yellow                                                     73 and gl3 1/2 le med. blue,                                                   moderate blue 182                                         Glucose     Growth:  good, plicate, no aerial                                  Yeast Extract        mycelium, no diffusible                                   Agar                 pigment                                                               Color:   g3nc amber, deep orange                                                        yellow 69                                                 Potato Slice                                                                               Growth:  good in the presence of CaCO.sub.3                                             fair in the absence of CaCO.sub.3                         Sucrose     Growth:  fair to poor, granular, no                                Nitrate Agar         aerial mycelium, no diffusible                            (Czapek's Agar)      pigment                                                               Color:   g41a orange, strong orange 50                             Tyrosine Agar                                                                  a) Tyrosine Yeast                                                                          Growth:  good, crystals poorly dissol-                             extract              ved, no diffusible pigment                                b) Tyrosine beef                                                                           Growth:  fair, crystals poorly dissol-                             extract              ved, faint brown diffusible                                                    pigment produced                                          Observations at 2,                                                             7 & 14 days (after                                                             Gordon & Smith J.                                                              Bact. 69, 147)                                                                 Peptone Iron Agar                                                                          Growth:  fair to poor, no reaction                                 Observations at 2,                                                             7 & 14 days                                                                    Bromo cresol         Milk not peptonized, pH                                   purple milk          unchanged                                                 ______________________________________                                          * NZ Amine - a peptone resulting from the enzymatic digestion of casein        and serving as a source of nitrogen?                                     

                  TABLE II                                                         ______________________________________                                         Carbohydrate Utilization                                                       Carbohydrate              Observation                                          ______________________________________                                         L-Arabinose               poor growth                                          Cellulose                 poor growth                                          D-Glucose                 good growth                                          D-Galactose               good growth                                          β-Lactose            fair growth                                          D-Levulose                good growth                                          D-Mannose                 good growth                                          D-Raffinose               poor growth                                          L-Rhamnose                good growth                                          Starch                    fair growth                                          Sucrose                   good growth                                          D-Xylose                  poor growth                                          Inositol                  good growth                                          D-Mannitol                good growth                                          Sorbitol                  poor growth                                          Control                                                                        0.5% yeast extract        poor growth                                          ______________________________________                                    

                  TABLE III                                                        ______________________________________                                         Nitrogen Utilization                                                           Nitrogen                                                                       Source         Observation                                                     ______________________________________                                         0.5% Yeast                                                                               Growth:  good, raised, folded, no                                    Extinct            aerial mycelium, no diffusible                              (Difco)*           pigment                                                               Color:   g31c amber, deep orange yellow                                                 69                                                          1.0% NZ   Growth:  good, no aerial mycelium, dark                              Amine **           brown diffusible pigment some-                              Type A             times produced                                                        Color:   g4pe orange rust, deep orange                                                  51                                                          1%        Growth:  poor, flat, no aerial mycelium,                             Asparasine         no diffusible pigment                                                 Color:   g18pn dark teal blue, dark                                                     greenish blue 174                                           1% Glutamic                                                                              Growth:  poor, flat, no aerial mycelium,                             Acid               no diffusible pigment                                                 Color:   g18pn dark teal blue, dark                                                     greenish blue 174                                           1% Sodium Growth:  very poor, not recordable                                   Nitrate                                                                        1% Ammonium                                                                              Growth:  very poor, not recordable                                   Nitrate                                                                        ______________________________________                                          * "Difco" is a registered trade mark (Difco Laboratories Inc., Detroit,        Michigan, U.S.A.)?                                                             ** NZ Amine - a peptone resulting from the anzymatic digestion of casein       and serving as a source of nitrogen?                                     

                                      TABLE IV                                     __________________________________________________________________________     Growth Characteristics of Actinoplanes Species                                       A.caeruleus                                                                            A.philippinensis                                                                         A.brasiliensis                                                                          A.missouriensis                                                                         A.utahensis                                                                           Actinoplanes                                                                           A.italacus            Medium                                                                               NRRL 5325                                                                              ATCC 12427                                                                               ATCC25844                                                                               CBS 188.64                                                                              CBS 367.66                                                                            sp.NRRL                                                                                ATCC                  __________________________________________________________________________                                                              27366                 Bennett's                                                                      Agar G                                                                               good    good      heavy    heavy    good   good    abundant               S    plicate slightly wrinkled                                                                        thick and                                                                               smooth   wrinkled and                                                                          N.D.    wrinkled                                      wrinkled          glistening                            C    strong yellow                                                                          yellow ochre                                                                             deep orange                                                                             pale orange                                                                             orange orange  light amber                 brown, areas                                       brown                       of dark green-                                                                 ish black                                                                Glucose                                                                        Asparagine                                                                     Agar G                                                                               good    good      good     good     good   abundant                                                                               scarce                 S    plicate smooth    thin and smooth   smooth and                                                                            N.D.                                                  smooth            glistening                            C    moderate                                                                               orange    pale rose                                                                               deep orange                                                                             deep orange                                                                           pale orange                                                                            light                       orange                                             cherry                Czapek-Dox                                                                     Agar G                                                                               fair to poor                                                                           good      moderate moderate moderate                                                                              abundant                                                                               N.D.                   S    granular                                                                               slightly wrinkled                                                                        thin, wrinkled                                                                          smooth   smooth N.D.                           C    strong orange                                                                          yellow ochre                                                                             deep orange                                                                             straw    straw  medium orange                 Nutrient                                                                       Agar G                                                                               fair    moderate  moderate good     good   scant   abundant               S    granular                                                                               smooth and thin                                                                          thin and smooth                                                                         thick and                                                                               smooth         smooth                                                 wrinkled                                       C    deep orange                                                                            pale orange                                                                              pale orange rose                                                                        deep orange                                                                             orange pale orange                                                                            deep amber                  yellow speck-                                                                  led with dark                                                                  greenish blue                                                            Potato                                                                         Agar G                                                                               fair to moderate  good     moderate moderate                                                                              N.D.    abundant                    moderate                                                                  S    flat,   smooth    smooth   thin and smooth and     smooth                      membranous                 wrinkled thin                                  C    deep orange                                                                            yellow brown                                                                             rose orange                                                                             pale orange                                                                             pale orange    light cherry                yellow                                             with amber                                                                     edge                  Calcium                                                                        Maleate G                                                                            very poor                                                                              moderate  good     moderate moderate                                                                              very weak                                                                              scarce                 S    non-character-                                                                         smooth    smooth   thin and smooth and                                 isable                     wrinkled thin                                  C    non-character-                                                                         yellow brown                                                                             rose orange                                                                             pale orange                                                                             pale orange    pale orange                 isable                                                                   __________________________________________________________________________      KEY:                                                                           G = Growth                                                                     S = Surface characteristics                                                    C = Colony color                                                         

                                      TABLE V                                      __________________________________________________________________________     Physiological Characteristics                                                            A.caeruleus                                                                           A.philippinensis                                                                        A.brasiliensis                                                                         A.missouriensis                              Test      NRRL 5325                                                                             ATCC 12427                                                                              ATCC 25844                                                                             CBS 188.64                                   __________________________________________________________________________     Starch    ++     +++      +++     +                                            Hydrolysis                                                                     H.sub.2 S -      +/-      -       -                                            Formation                                                                      Melanin   +      -        -       -                                            Tyrosine  +      +        -       +++                                          Hydrolysis                                                                     Xanthine  N.D.   -        -       -                                            Hydrolysis                                                                     Casein    +      +        +       +                                            Hydrolysis                                                                     Calcium Maleate                                                                          -      +        +       +/-                                          Hydrolysis                                                                     Nitrate   +      +        +       +                                            Reduction                                                                      Litmus    N.C.   N.C.     N.C.    N.C.                                         Milk      N.P.   N.P.     N.P.    N.P.                                         Gelatin   +      +        +       -                                            Liquification                                                                            A.caeruleus                                                                           A.utahensis                                                                           Actinoplanes                                                                           A.italacus                                                                            A.armeniacus                            Test      NRRL 5325                                                                             CBS 367.66                                                                            sp.NRRL 3884                                                                           ATCC 27366                                                                            ATCC 15676                              __________________________________________________________________________     Starch    ++     +      N.D.    +/-    ++                                      Hydrolysis                                                                     H.sub.2 S -      +      N.D.    +      +                                       Formation                                                                      Melanin   +      +      +       +      +                                       Tyrosine  +      +/-    N.D.    +      ++                                      Hydrolysis                                                                     Xanthine  N.D.   -      N.D.    N.D.   N.D.                                    Hydrolysis                                                                     Casein    +      +      N.D.    +      +++                                     Hydrolysis                                                                     Calcium Maleate                                                                          -      +      N.D.    -      +/-                                     Hydrolysis                                                                     Nitrate   +      +      +       +/-    +                                       Reduction                                                                      Litmus    N.C.   N.C.   N.C.    N.C.   N.C.                                    Milk      N.P.   N.P.   N.P.    Peptonised                                                                            Peptonised                              Gelatin   +      +/-    +       +      +                                       Liquification                                                                  __________________________________________________________________________     KEY:                                                                               -  = negative response                                                                               N.D. = not determined                                    +/ = doubtful response                                                                               N.C. = no coagulation                                    +  = weak positive response                                                                          N.P. = no peptonization                                  ++ = positive response                                                         +++                                                                               = strong positive response                                         

                                      TABLE VI                                     __________________________________________________________________________     Comparison of carbohydrate utilization of A. caeruleus                         with other Actinoplanes species                                                Carbo-                                                                               A.caeruleus                                                                           A.philippinensis                                                                        A.brasiliensis                                                                         A.missouriensis                                  hydrate                                                                              NRRL 5325                                                                             ATCC 12427                                                                              ATCC 25844                                                                             CBS 188.64                                       __________________________________________________________________________     Inositol                                                                             ++     ++       +       -                                                Fructose                                                                             ++     ++       ++      ++                                               Rhamnose                                                                             ++     ++       ++      ++                                               Mannitol                                                                             ++     ++       ++      ++                                               Xylose                                                                               -      ++       ++      ++                                               Raffinose                                                                            -      ++       -       -                                                Arabinose                                                                            -      ++       ++      ++                                               Cellulose                                                                            +/-    +        +       -                                                Sucrose                                                                              ++     ++       ++      ++                                               Glucose                                                                              ++     ++       ++      ++                                               Control                                                                              -      -        -       -                                                Carbo-                                                                               A.caeruleus                                                                           A.utahensis                                                                             A.Actinoplanes                                                                         A.italacus                                                                            A.armeniacus                              hydrate                                                                              NRRL 5325                                                                             CBS 367.66                                                                              sp.NRRL 3884                                                                           ATCC 27366                                                                            ATCC 15676                                __________________________________________________________________________     Inositol                                                                             ++     -        -       ++     +/-                                       Fructose                                                                             ++     ++       ++      ++     ++                                        Rhamnose                                                                             ++     ++       +/-     ++     ++                                        Mannitol                                                                             ++     ++       ++      ++     ++                                        Xylose                                                                               -      ++       ++      ++     ++                                        Raffinose                                                                            -      -        -       -      -                                         Arabinose                                                                            -      ++       ++      ++     ++                                        Cellulose                                                                            +/-    -        -       -      N.D.                                      Sucrose                                                                              ++     ++       +/-     ++     -                                         Glucose                                                                              ++     ++       ++      ++     ++                                        Control                                                                              -      -        N.D.    -      -                                         __________________________________________________________________________      Key                                                                            - = no utilization                                                             +/- = slightly better than control                                             + = fiar to moderate utilization                                               ++ = strong utilization                                                        N.D. = not determined                                                    

Fermentation of the Microorganism

Fermentation, or cultivation, of the microorganism is usually effected in two stages, the first being an inoculum production stage and the second the antibiotic production stage.

Production of the inoculum is typically effected by cultivation in a suitable nutrient medium of a culture from an agar slant of Actinoplanes caeruleus.

Antibiotic production is typically effected by cultivation of a sample of the inoculum in a suitable aqueous nutrient medium under submerged aerobic conditions at a temperature of about 23° to 40° C., preferably at 28° C., and at a pH of from about 6.5 to 8.0, with agitation, until substantial antibiotic activity is imparted to the medium. A fermentation time of from about 3 to 7 days may suitably be employed. To determine when peak antibiotic production has been reached, samples of the medium may be periodically assayed for antibiotic content as hereinafter described. In this manner we have found in specific instances that a preferred cultivation period is about 4 days.

As nutrient medium there may be employed any suitable medium containing a source of carbon, for example an assimilable carbohydrate, and a source of nitrogen, for example an assimilable nitrogenous or proteinaceous material.

Preferred carbon sources include glucose, brown sugar, sucrose glycerol, starch, corn starch, lactose, dextrin, molasses, while preferred nitrogen sources include corn steep liquor, autolyzed brewer's yeast, soyabean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distiller's solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be advantageously used. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not usually be added to the fermentation media since tap water and the unpurified ingredients used as media components generally contains such metals.

Where only small amounts of antibiotic are required then in place of submerged cultivation, surface cultivation may be employed.

In a method employed herein for determining when peak antibiotic production has occurred, a sample of a specified volume of the fermentation medium is taken, the antibiotic extracted therefrom and then assayed using a standard cylinder cup assay method. Extraction of the antibiotic from the sample may be effected using a water-immiscible organic solvent such as chloroform, toluene, benzene, diethylene glycol diethyl ether or the like. Alternatively, an organic solvent sparingly soluble in water may be employed, for example n-butanol. A sample of fermentation medium may typically contain on a weight/volume basis 175 to 1000 mcg/ml of crude antibiotic although in high-yielding fermentations, concentrations several times higher than the quoted upper limit may be obtained. The obtained solvent is evaporated to dryness in vacuo, the residue weighed and dissolved in dimethylformamide to give a solution having an estimated antibiotic 67-121 concentration of approximately 1000 mcg/ml. An aliquot of this solution is then taken and diluted with 0.1 molar phosphate buffer of pH 8.0 to give a solution having an estimated antibiotic 67-121 concentration of approximately 3 mcg/ml.

The precise concentration of antibiotic 67-121 in the latter solution is then determined using a standard cylinder cup assay with Saccharomyces cervisiae ATCC 9763 as the test organism and employing as standard a pure sample of antibiotic 67-121 complex which is assigned a potency of 1000 mcg/mg.

The Antibiotic 67-121 reference standard employed has a potency of 4000 mcg/mg, expressed as candicidin, in the candicidin assay set forth in Title 21 of the Code of Federal Regulation, Section 148v.

Isolation and Purification of the Antibiotic Product

Following peak production, the polyene antibiotic product is isolated from the fermented medium using methods known per se for polyene antibiotics.

Thus, for example, a method using the following sequence of steps may be employed:

(a) acidify the whole broth to a pH of 2.5 to 4.0;

(b) filter off the mycelium from the acidified broth;

(c) extract the mycelium one or more times with a suitable water immiscible or sparingly miscible organic solvent;

(d) combine the organic solvent extracts and evaporate to a residue;

(e) triturate the residue with a non-polar organic solvent such as a saturated or unsaturated hydrocarbon or an alkyl ether, preferably diethyl ether;

(f) filter off the resulting suspension which represents the crude antibiotic 67-121 complex.

Alternatively, the polyene antibiotic may be isolated by acidifying the whole broth to a pH of 2.5 to 4.0, directly extracting the acidified broth with a suitable water immiscible or sparingly miscible organic solvent and then subjecting the combined extracts to steps (d) to (f) above.

In use, we have found that by the above specified methods a crude antibiotic 67-121 complex assaying in the previously described cylinder cup assay at 250 mcg/mg. can be obtained.

Purification of the crude polyene antibiotic complex may be effected by methods known in the art, such as fractional crystallisation, sequential dissolution and re-precipitation, counter-current extraction techniques and the like.

Thus, a crude antibiotic 67-121 product assaying at 250 mcg/mg. may be purified by dissolving this product in the lower phase of a chloroform:methanol:water mixture having a volume ratio of 2:2:1.

The resulting solution is filtered to remove insoluble material and the filtrate is then added to diethyl ether (10 volumes diethyl ether to 1 volume of filtrate) and the resulting yellow precipitate filtered off. The precipitate is then dissolved in an adequate amount of the upper phase of the aforementioned chloroform:methanol:water mixture, filtered and the filtrate allowed to stand in vacuo until sufficient solvent has evaporated to result in precipitation of the purified antibiotic complex. The precipitated purified antibiotic complex is filtered off, washed with diethyl ether and then dried in vacuo. The purified antibiotic 67-121 complex obtained in this manner assays at 1000 mcg/mg. as antibiotic 67-121 complex (or at 4000 mcg/mg. in terms of candicidin).

Separation of the Antibiotic Components

Suitably the antibiotic 67-121 complex may be separated into its components by chromotographic methods, such as by column chromatography. Specifically in employing column chromatography silica gel may be employed as the column material and the lower phase of a chloroform:methanol:water mixture (2:2:1 parts by volume) as the eluant.

If desired, prior to the above chromatographic separation step, a partial separation may be effected by dissolving the 67-121 complex in the lower phase of a chloroform:methanol:water mixture (2:2:1 parts by volume) and then effecting partial precipitation of the dissolved material by addition of methanol.

Column chromatographic separation of the Antibiotic 67-121 complex has shown the complex to comprise four component antibiotics herein designated Antibiotics 67-121A, 67-121B, 67-121C and 67-121D, this designation following the order in which the antibiotics emerge from the column. Using silica gel plates and the above specified solvent system, the determined Rf values for the individual components were antibiotic 67-121A= 0.17, 67-121B= 0.14, 67-121C= 0.11 and 67-121D= 0.07. Elution of the antibiotic components from the column may be readily monitored on silica gel thin-layer plates using the solvent mixture employed in the column chromatographic separation, the presence of antibiotic material on the plate being identified by any convenient means such as by bioautography or alternatively by spraying the plate with say a sulfuric and methanol mixture and then heating the plate to reveal the presence of antibiotic material.

The relative proportion of the antibiotic components may well vary from one fermentation to another, but in general it may be said that Antibiotic 67-121C will usually be the major component and 67-121D a trace component.

By way of illustration, it may be mentioned that in a specific example the relative proportions of components on a weight basis were 67-121A 12%, 67-121B 12%, 67-121C 76% and 67-121D trace.

The Antibiotic Products

As previously stated the Antibiotic 67-121 complex comprises the component antibiotics designated 67-121A, 671-121B, 671-121C and 671-121D, and these antibiotics have been classified as polyene macrolide antibiotics. Further, from their characteristic ultraviolet absorption spectra and infrared absorption spectra the antibiotics have been classified as heptaene macrolide antibiotics.

Chemical and physical data for the antibiotic 67-121 complex and its components are set forth below.

Chemical and Physical Properties of Antibiotic 67-121 Complex.

Table 1 below sets forth ultraviolet absorption spectral data for a sample of Antibiotic 67-121 complex and, for comparison purposes, corresponding data taken from the literature for a number of other heptaene antibiotics. Table 2 correspondingly sets forth data for elemental analysis, optical rotation, melting point and also lists R_(f) values. These R_(f) values were obtained with a solvent system consisting of a mixture of n-butanol (6 parts), acetic acid (2 parts), water (2 parts) and dioxane (1 part) and detection was made either by bioautography against Candida albicans or by spraying the chromatographic plate with a sulfuric acid, methanol spray.

In Table 3 certain stability data for the Antibiotic 67-121 complex are presented, the antibiotic being heated with the appropriate 0.1M buffer for the stated periods of time on a boiling water bath. Specifically, a borate buffer was employed for pH values 9.0 to 10.0 and for the range 2.2 to 8.0 buffers marketed under the trade designation McIlvaines buffer were employed. The stability was ascertained by determining the antifungal activity, after heating for the specified time, using a disc assay method (6.25 mm discs) with Candida albicans as the test organism. The results are expressed in terms of the diameter of the zone of inhibition given in mm. From Table 3 it will be seen that the Antibiotic 67-121 complex is fairly stable at 100° C. in the pH range of 7.0 to 9.0. At pH 10 it is somewhat less stable, the activity decreasing rapidly after 15 minutes at 100° C. Below pH 5.0 the antibiotic is relatively unstable.

As will be seen from Table 2, the Antibiotic 67-121 complex can be distinguished chromatographically from the other specified antibiotics with the exception of candidin. Antibiotic 67-121 can be distinguished from candidin, however, on the basis its elemental analysis and optical rotation (for the latter see the values given thereinafter for the components B, C and D).

To further characterise the Antibiotic 67-121 complex, a standard classification procedure for heptaene antibiotics was employed (see Tetrahedron 25 pp 2229-2232). Acid hydrolysis yielded an amino sugar moiety and, in addition, potassium hydroxide treatment yielded an aromatic ketone moiety, the antibiotic product being accordingly placed in the heptaene subgroup II classification. The heptaene subgroup II includes such known antibiotics as candicidin, trichomycin B and perimycin.

                  Table 1                                                          ______________________________________                                         Ultra violet absorption data                                                   Maxima and in brackets E.sub.1cm.sup.1% values.                                           Maxima in mμ                                                     Antibiotic   in MeOH        in EtOH                                            ______________________________________                                         Antibiotic                                                                               342(340, 363(475),                                                                            344(385) 362(490),                                    67-121 complex                                                                           382(667), 403(605)                                                                            382(690) 404(625)                                     Ascosin "A"              340, 358, 377, 399                                    Ascosin "B"              340, 358, 376, 398                                    Candicidin               360, 380, 403                                         Candidin  363(1000), 382(1650)                                                                          364(985), 385(1730),                                            406(1860)      407(1910)                                             Candimycin                                                                               362, 382, 406                                                        Fungimycin               358, 376, 399                                         Hamycin   230, 285, 345, 363                                                             383(962), 406(918)                                                   Trichomycin                                                                              361.5(625, 382(863)                                                                           286, 346, 364, 384, 405                               "A"       404(750)       (Trichomycin complex)                                 Trichomycin                                                                              358, 377, 400                                                        "D"                                                                            ______________________________________                                    

                  TABLE 2                                                          ______________________________________                                                                 Optical  M.P.                                          Antibiotic                                                                               Elemental Analysis                                                                           Rotation ° C.                                                                           R.sub.f                                ______________________________________                                                 C     H      N                                                         Antibiotic                                                                     67-121 Complex                                                                           56.14   7.53   1.79        175    0.43                               Ascosin "A"                                                                    plus                          +13°*  0.53                               Ascosin "B"                                                                    Candicidin                                                                               63.29   7.16   2.33               0.54                               Candidin  60.06   9.65   1.65 +363°**                                                                        180    0.45                               Candimycin                                                                               57.17   8.81   1.70        230    0.53                               Fungimycin                                  0.54                               Hamycin   59.5    8.3    2.2  +216°***                                                                       160    0.52                                                                    (no                                                                            defi-                                                                          nite                                                                           m.p.)                                     Trichomycin                                                                              60.06   7.62   2.28        320    0.55                               "A"                                  (decompo-                                                                      sition)                                   Trichomycin                                                                              59.49   8.09   2.16                                                  "B"                                                                            ______________________________________                                           * in dimethylformamide; literature source does not specify concentration       ** 0.3 % in dimethylformamide                                                 *** 0.1 % in pyridine                                                    

                  Table 3                                                          ______________________________________                                         Heat and pH Stability of Antibiotic 67-121 complex                             (Expressed in terms of Antifungal Activity)                                    Time of Exposure at 100° C.                                             Results expressed in terms diameter of zone of inhibition                      in mmm                                                                         ______________________________________                                         pH      0 min.    5 min.    15 min.   30 min                                   ______________________________________                                         2.2     0         0         0         0                                        3.0     0         0         0         0                                        4.0     12        9         0         0                                        5.0     14        14        13        11                                       6.0     15        15        15        11                                       7.0     15        14        14        14                                       8.0     17        16        15        13                                       9.0     17        16        13        12                                       10.0    16        15        11        0                                        ______________________________________                                    

Chemical and Physical Properties of the Antibiotic 67-121 Components

    ______________________________________                                         Antibiotic 67-121A                                                             Colour and form - Yellow needles                                               m.p. = 185° - 190° C. (Decomposition)                            Elemental Analysis                                                             C = 59.40%                                                                     H = 7.90%                                                                      N = 2.60%                                                                      M.Wt. =     1218 (osmometry)                                                   [α].sub.D.sup.26                                                               =     +161.6° (0.3% in dimethylformamide)                         pka   =     6.0 (COO.sup.-)                                                                9.0 (NH.sub.3 .sup.+(66% aqueous dimethyl formamide)               U.V. data                                                                              (in tetrahydrofuran:water, ratio 4:1)                                  λmax                                                                            408 mμ (E.sub.1cm.sup.1% = 950)                                             384 mμ (E.sub.1cm.sup.1% = 1100)                                            363 mμ (E.sub.1cm.sup.1% = 680)                                     I.R. data                                                                              (in Nujol)                                                             νmax 3350, 1725 and 1665 cm.sup.-.sup.1                                     NMR data                                                                               (in hexa-deutero-dimethylsulfoxide)                                    δ 2.73 ppm (3H, singlet, aromatic--N--CH.sub.3)                                  6.55 ppm (2H, doublet, J = 8.5 Herz., aromatic H)                              7.64 ppm (2H, doublet, J = 8.5 Herz., aromatic H)                      Antibiotic 67-121B                                                             Colour and form - Yellow needles                                               m.p. = 175° - 182° C. (Decomposition)                            Elemental Analysis                                                             C =  59.70%                                                                    H = 7.65%                                                                      N = 2.17%                                                                      M.Wt. =     1218 (osmometry)                                                   [α].sub.D.sup.26                                                               =     +199.7° (0.3% in dimethylformamide)                         pka   =     6.3 (COO.sup.-)                                                                9.0 (NH.sub.3.sup.+) (66% aqueous dimethylformamide)               U.V. data                                                                              (in tetrahydrofuran: water, ratio 4:1)                                 λ max                                                                           408 mμ (E.sub.1cm.sup.1% = 620)                                             383 mμ (E.sub.1cm.sup.1% = 750)                                             362 mμ (E.sub.1cm.sup.1% = 490)                                     I.R.data                                                                               (in Nujol)                                                             ν max                                                                               3350, 1725 and 1665 cm.sup.-.sup.1                                     NMR data                                                                               (in hexa-deutero-dimethylsulfoxide)                                    δ 6.58 ppm (2H, doublet, J = 8.5 Herz., aromatic H)                              7.60 ppm (2H, doublet, J = 8.5 Herz, aromatic H)                       Antibiotic 67-121C                                                             Colour and form - Yellow needles                                               m.p. = 175° C. - 180° C. (Decomposition)                         Elemental Analysis                                                             C = 57.06%                                                                     H = 7.30%                                                                      N = 2.07%                                                                      M.Wt. =     1266 (osmometry)                                                   [α].sub.D.sup.26                                                               =     +143.8° (0.3% in dimethylformamide)                         pka   =     6.0 (COO.sup.-)                                                                9.0 (NH.sub.3 .sup.+) (66% in aqueous dimethylformamide)           U.V. data                                                                              (in tetrahyrofuran:water, ratio 4:1)                                   λ max                                                                           408 mμ (E.sub.1cm.sup.1% = 800)                                     383 mμ (E.sub.1cm.sup.1% = 910)                                                     362 mμ (E.sub.1cm.sup.1% = 600)                                     I.R. data                                                                              (in Nujol)                                                             ν max                                                                               3350, 1725 and 1665 cm.sup.-.sup.1                                     NMR data                                                                               (in hexa-deutero-dimethylsulfoxide)                                    δ 2.76 ppm (3H, singlet, aromatic N--CH.sub.3)                                   6.57 pm (2H, doublet, J = 8.5 Herz., aromatic H)                               7.65 ppm (2H, doublet, J = 8.5 Herz., aromatic H)                      ______________________________________                                    

As for the antibiotic 67-121 complex, the individual component antibiotics from their characteristic ultraviolet and infra-red absorption spectra are classified as heptaene macrolide antibiotics having numerous hydroxyl groups attached to the macrolide ring. Further, the individual components are classified as heptaene subgroup II antibiotics (see the hereinbefore specified classification). Degradation studies, as well as nuclear magnetic resonance and mass spectrometric data, show that the individual components contain the aromatic ketonic moiety: ##STR1## in which, for antibiotics 67-121 A and C, R is methyl and for Antibiotic 67-121 B, R is hydrogen.

Acetolysis of antibiotics 67-121A and 67-121B reveals the presence of a D-mycosamine sugar unit glycosidically attached to an allylic hydroxy group of the aglycone.

Acetolysis of antibiotic 67-121C reveals the presence of a novel disaccheride unit attached to an allylic hydroxy group of the aglycone, the disaccheride being indicated as [O-β-D-mannopyranosyl (1→4)-D-mycosamine] of the formula ##STR2## wherein the wavy line denotes that the stereochemistry of the glycosidic linkage of the disaccharide to the aglycone has not yet been established.

The antibiotic components contain a carboxylic acid group which is amenable to salt and ester formation by methods known per se. Thus for example we have obtained corresponding esters using a diazomethane route, these esters also being found to possess antifungal activity. The antibiotic and esters thereof form acid addition salts with the appropriate acids for example mineral acids such as hydrochloric acid, sulfuric acid and phosphoric acid or organic acids such as acetic acid, citric acid, tartaric acid and the like.

BIOLOGICAL ACTIVITY OF THE ANTIBIOTIC PRODUCTS (A) In Vitro Antifungal Activity

In vitro antifungal activity data for the antibiotic 67-121 complex are set forth in Table 4. For reference, data on candicidin are also given. The quoted minimal inhibitory concentrations (MIC) were obtained by conventional tube dilution techniques using Sabouraud's dextrose broth.

As representative of the component antibiotics, antibiotic 67-121A and antibiotic 67-121C were tested in the same manner as the complex. Antibiotic 67-121A gave MIC values of from less than 0.01 up to 0.03 mcg/ml. against 18 strains of Candida albicans, while antibiotic 67-121C gave MIC values of 0.08 to 0.3 mcg/ml. against 9 strains of Candida albicans after incubation times of 48 hours. Further testing of antibiotic 67-121C under the same conditions gave MIC values of 0.3 mcg/ml. against two strains of Torulopsis and a single strain of Cryptococcus. Strains of Aspergillus and Dermatophytes were generally less sensitive with MIC values for antibiotic 67-121C of from 3 to 25 mcg/ml.

                  Table 4                                                          ______________________________________                                         Antibiotic 67-121 complex - In vitro antifungal activity                       Minimum Inhibitory Concentration (M.I.C.)                                      in mcg/ml                                                                                67-121 complex                                                                             Candicidin                                               Organism    24 hrs.  48 hrs.  24 hrs.                                                                               48 hrs.                                   ______________________________________                                         Candida 406     0.03     0.3    0.03   0.08                                     albicans                                                                              402     0.03     0.3    0.03   0.08                                            420     3.0      3.0    3.0    7.5                                             404     0.8      3.0    0.8    3.0                                             403     0.03     0.3    0.03   0.08                                            401     0.8      0.8    0.8    3.0                                             1       0.3      0.3                                                           12031   0.3      0.3                                                           Flo     0.3      0.3                                                           404A    0.3      0.3                                                   Torulopsis                                                                             1       0.3      0.8    0.03   0.3                                      glabrata                                                                              2       0.3      0.8    0.03   0.08                                            3       0.3      0.8    0.03   0.08                                            12      0.8      0.8                                                           16      0.3      0.8    0.03   0.3                                             17      0.3      0.3    0.03   0.08                                            24      0.8      0.8                                                           27      0.8      3.0                                                           34      0.3      0.3                                                           76      0.3      0.3                                                           46      0.3      0.3                                                             67-121 complex                                                                             Candicidin                                                         72 hrs.                                                              Aspergillus niger       7.5    7.5                                             Trichophyton mentagrophytes                                                                     17     7.5    25                                              Trichophyton mentagrophytes                                                                     1A     3.0    17.5                                            Trichophyton soudanese                                                                          2      7.5    25                                              Trichophyton - 16 strains                                                       (12 species)           5.0    5.0                                             Microsporum - 6 strains                                                         (6 species)            5.0                                                    Microsporum distortum   3.0                                                    Phialophora (2 strains) 5.0                                                    ______________________________________                                    

(B) In Vivo Antifungal Activity

The in vivo protective activity of the antibiotic products in mice (Carworth farm CF 1 mice, each weighing about 20 g.) against Candida albicans was determined. The antibiotic was administered by the stated route in an aqueous vehicle containing 0.5% carboxymethyl cellulose and in two doses. One dose was administered shortly before intravenous infection with a lethal dose of Candida albicans and the other dose was administered 4 hours after infection. Most of the unprotected control mice died within 24 to 48 hours after infection. The survival rate was determined at the end of 24 and 48 hours and from this using standard procedures the PD₅₀ values were determined.

The results obtained for the Antibiotic 67-121 complex are given in Table 5 together with data for candicidin (for comparison purposes). The PD₅₀ values were determined on survivors 48 hours after infection.

                  Table 5                                                          ______________________________________                                                                Controls (%                                                     PD.sub.50 (mg/kg)                                                                             survivors)                                                            Antibiotic   Candi-                                                                               24       48                                   Test Route    67-121 complex                                                                              cidin hrs.     hrs.                                 ______________________________________                                         1    Subcu-   2.5          5     20       20                                        taneous                                                                        Oral     2.5          25                                                  2    Subcu-   5.0          5     50       16                                        taneous                                                                        Oral     2.5          5                                                   3    Subcu-   15           25    0        0                                         taneous                                                                   4    Subcu-   1.5          2.5   20       20                                        taneous                                                                        Oral     1.5          40                                                  ______________________________________                                    

The PD₅₀ values for antibiotics 67-121A, 67-121B and 67-121C using CF 1 mice were determined using essentially the above described method, with the exception that the antibiotic was administered as a single dose one hour after infection with Candida albicans.

The PD₅₀ values were determined on survivors 72 hours after infection. Of the untreated, infected control mice, 80% died within 24 hours and 100% within 48 hours. The results are given in Table 6.

                  Table 6                                                          ______________________________________                                         In vivo antifungal activity                                                                     PD.sub.50 (mg/kg)                                                        Antibiotic  Antibiotic  Antibiotic                                  Route      67-121A     67-121B     67-121C                                     ______________________________________                                         Subcutaneous                                                                              2.5         15.0        2.1                                         Oral       1.5         18.0        1.5                                         ______________________________________                                    

(C) Acute Toxicity Data

The acute toxicity of the antibiotic 67-121 complex in mice was determined via intraperitoneal (I.P.) and subcutaneous (S.C.) administration. For comparison, the values of candicidin were determined. The results are given in Table 7 below.

                  Table 7                                                          ______________________________________                                         LD.sub.50 (mg/kg)                                                                         Antibiotic 67-121                                                   Route      complex             Candicidin                                      ______________________________________                                         I.P.       5 - 15              8 - 22                                          S.C.       300                 300                                             ______________________________________                                    

Similarly, in another series of tests the acute toxicity of antibiotics 67-121A, 67-121B and 67-121C were determined in male CF 1 mice. The results are given in Table 8 below.

                  Table 8                                                          ______________________________________                                         LD.sub.50 (mg/kg)                                                                       Antibiotic   Antibiotic   Antibiotic                                  Route    67-121A      67- 121B     67-121C                                     ______________________________________                                         I.P.     2.4          6.0          6.0                                         S.C.     >50          >50          --                                          Oral     >50          >50          --                                          ______________________________________                                    

The novel antibiotic products may typically be administered topically, orally or parentally, in free form or in the form of a suitable derivative, and preferably in admixture with a pharmaceutically acceptable carrier or excipient.

The precise dose to be administered in any particular dosage form will depend upon such factors as the stage and severity of the infection, the susceptibility of the infecting organism to the antibiotic and the individual characteristics of the animal species being treated. It may be said, however, that for intravenous use of the antibiotics of this invention an amount of from about 0.25 mg. to about 1.5 mg. per kilogram of body weight per dose is indicated. For oral administration, a dose in the range of 5 to 50 mgs. would be indicated. In the foregoing, the individual dose would be administered one to four times a day. Topical preparations would usually be administered in the form of creams, ointments, powders or vaginal suppositories. Oral preparations would be in the form of elixirs, tablets, capsules and the like.

EXAMPLES

The invention will now be further illustrated by way of the following examples in which Example 1 illustrates the cultivation of Actinoplanes caeruleus (specifically, that assigned NRRL 5325 was employed) and production of antibiotic 67-121 complex, Examples 2 to 5 illustrate the isolation and purification of the 67-121 complex, Example 6 illustrates the resolution of the 67-121 complex into its component antibiotics, Examples 7 and 8 illustrate the preparation of specified derivatives of antibiotic 67-121C and the remaining Examples illustrate compositions containing specified antibiotic products.

EXAMPLE 1 Cultivation of Actinoplanes caeruleus and Production of Antibiotic 67-121 Complex. A. Inoculum Preparation

Add a loopful (e.g. about 0.5 ml.) of culture from an agar slant of Actinoplanes caeruleus to each of a series of 300 ml. Erlenmeyer flasks containing 100 ml. of a sterile medium having the following composition:

    ______________________________________                                         Beef extract         3.0     g.                                                Yeast extract        5.0     g.                                                Tryptose             5.0     g.                                                Soluble starch       24.0    g.                                                Dextrose             1.0     g.                                                Calcium carbonate    2.0     g.                                                Tap water            1000    ml.                                               ______________________________________                                    

Incubate the contents of the flasks with agitation on a rotary shaker at 250 to 300 rpm for about 48 hours at about 35° C.

B. Cultivation and Antibiotic Production

Transfer under aseptic conditions 5 ml. of the inoculum prepared in step A to each of a series of 500 ml. Erlenmeyer flasks containing 100 ml. of sterile medium having the following composition:

    ______________________________________                                         Soybean meal         30      g.                                                Dextrose             40      g.                                                Calcium carbonate    10      g.                                                Tap water            1000    ml.                                               ______________________________________                                    

Effect the submerged aerobic cultivation by fermenting the contents of the flasks at about 28° C. for 3 to 4 days with rotary agitation at about 300 rpm.

After about the first 24 hours, samples are periodically taken from the fermented media and assayed for antibiotic activity so that peak production, that is the stage at which the antibiotic activity of the fermented media reaches a peak can be ascertained. When fermentation is effectively complete, as evidenced by peak production, the contents of the flasks may be conveniently combined for subsequent isolation of the antibiotically active product.

Alternatively, the above fermentation may be carried out using other suitable media such as the following:

    ______________________________________                                         (a)     Yeast extract    5       g.                                                    Casein hydrolysate                                                                              5       g.                                                    Dextrose         10      g.                                                    Soluble starch   20      g.                                                    Calcium carbonate                                                                               4       g.                                                    Tap water        1000    ml.                                           (b)     Soybean meal     30      g.                                                    Corn steep dried                                                               solids           5       g.                                                    Starch           20      g.                                                    Dextrose         10      g.                                                    Calcium carbonate                                                                               7       g.                                                    Tap water        1000    ml.                                           ______________________________________                                    

EXAMPLE 2 Isolation of the Antibiotic 67-121 Complex

Acidify the fermented medium obtained from Example 1 with a mineral acid such as hydrochloric acid to a pH of from 2.5 to 4.0. Add a suitable filter aid (we specifically employed that sold under the trade designation Hyflo-supercel which is a free flowing diatomaceous earth marketed by Johns Manville Co. U.S.A.) and then extract the resulting filter cake repeatedly with n-butanol until the filter cake is substantially free of antibiotic material. Combine the n-butanol extracts and evaporate in vacuo to a residue. Triturate the residue with a mixture containing by volume 1 part of diethyl ether to 1 part of acetone, filter, re-triturate the residue with diethyl ether, again filter and then dry the resulting residue in vacuo. The resulting antibiotic-121 complex is generally a green-yellow powder assaying in the hereinbefore specified cylinder cup assay at 1000 mcg/mg. as candicidin or 250 mcg/mg. as antibiotic 67-121 complex.

EXAMPLE 3 Alternative Isolation Method for the Antibiotic 67-121 Complex

Treat the fermented medium as described in Example 2 to give a filter cake and then wash this cake with acetone to remove most of the water. Extract the filter cake with a mixture consisting of both phases of a chloroform:methanol:water mixture having a volume/volume ratio of 18:6:1 until the filter cake is substantially free of antibiotic. Combine the extracts, separate the liquid phases, discard the aqueous phase and then evaporate the organic phase to a residue of crude antibiotic 67-121 complex assaying at about 250 mcg/mg.

EXAMPLE 4 Further Isolation Method for the Antibiotic 67-121 Complex

Acidify one liter of the fermentation broth obtained from Example 1 with a mineral acid such as hydrochloric acid to a pH of about 3.5. Extract the acidified broth twice with two liter portions of butanol saturated with water. Evaporate the combined extracts to a residue and then dissolve the residue in the lower phase of a chloroform:methanol:water mixture having a volume ratio of 2:2:1. Dry the solution over anhydrous sodium sulfate and filter. Pour the dried solution into a diethyl ether:hexane mixture having a volume ratio of 6:4 while vigorously agitating the mixture. Filter and dry the resulting yellow-brown precipitate of crude antibiotic 67-121 complex (assaying at about 250 mcg/mg.). We have obtained a yield of 940 mg. of product.

EXAMPLE 5 Purification of the Antibiotic Complex

Dissolve 3.9 g. (obtained as described in Example 2) in the lower phase of a chloroform:methanol:water mixture (2:2:1). Filter off the insolubles and then add the filtrate to anhydrous diethyl ether. Filter the resulting yellow precipitate, wash several times with diethyl ethyl, and redissolve in the upper phase of the same system. Filter off the insolubles under vacuum. During the filtration a precipitate will form in the filtrate which is then filtered off, washed 3 to 4 times with diethyl ether and dried under vacuum. A yield of 410 mg. is obtained, the material having a potency of about 1000 mcg/mg as antibiotic 67-121.

EXAMPLE 6 Separation of the Antibiotic Complex into its Components

To a 5 cm. diameter column add 800 g. of chromatographic silica gel then, using as eluant the lower phase of a freshly prepared mixture of chloroform:methanol:water (2:2:1 ratio by volume), elute 4.6 g. of the antibiotic 67-121 complex obtained as for example by the method of Example 3.

Monitor the emergence of the antibiotic components by sampling the fractions collected and subjecting the samples to thin-layer chromatography or other suitable technique. Combine the eluate fractions containing each antibiotic fraction and concentrate to give the individual antibiotic fractions.

    ______________________________________                                         Antibiotic                      [α.sub.D.sup.26 (0.3%                    component                                                                              R.sub.f                                                                               Weight   m.p.° C.                                                                        dimethyl formamide)                            ______________________________________                                         67-121A 0.17   0.262g.  185-190*                                                                               +161.6                                         67-121B 0.14   0.260g.  175-182*                                                                               +199.7                                         67-14 121C                                                                             0.11   1.640g.  175-180*                                                                               +143.8                                         67-121D 0.07   trace    --      --                                             ______________________________________                                          (* - decomposition)                                                      

EXAMPLE 7 Preparation of Antibiotic 67-121C Methyl Ester

Dissolve 5 g. of antibiotic 67-121C in 150 ml. of dimethyl sulphoxide and 15 ml. of methanol. Cool the solution to about 5° C. and add 0.5 g. of diazomethane in 30 ml. of tetrahydrofuran. Allow the reaction to continue for 10 minutes then pour the solution into 10 volumes of vigorously agitated diethyl ether. Collect the product by filtration or centrifugation and wash with fresh diethyl ether. Dissolve the precipitate in the lower phase of a chloroform, methanol, water mixture (2:2:1 v/v), chromatograph on 500 g. of silica gel using the same solvent mixture, and obtain thereby 1.1 g of the title compound, having the following characteristics:

m.p.= 180°-183° C.;

elemental analysis: C= 56.30%, H= 7.21%, N= 1.84%;

λ max. (in tetrahydrofuran, water mixture 4:1 parts by volume) 409 mμ (E_(1cm) ^(1%) = 650), 385 mμ (E_(1cm) ^(1%) = 770), 364 mμ (E_(1cm) ^(1%) = 515);

δ(hexa-deutero-dimethyl sulfoxide) 3.4 ppm (3 H, singlet, COOCH₃).

By substituting an equivalent quantity of antibiotic 67-121A or antibiotic 67-121B for antibiotic 67-121C in the foregoing example and by following the procedure of said example, the methyl esters of the respective antibiotics may be prepared.

Similarly, other esterifying agents may be employed to form other lower alkyl esters such as ethyl, propyl, isopropyl, butyl or the like.

EXAMPLE 8 Preparation of Antibiotic 67-121C Methyl Ester Hydrochloride

Dissolve 1.3 g. of Antibiotic 67-121C methyl ester (prepared as described in Example 7) in a mixture of tetrahydrofuran and water (4:1 v/v) and cool to about 10° C. Add 10 ml. of 0.1 N hydrochloric acid to the solution, concentrate to a residue, redissolve in water and lyophilize to obtain 1.1 g. of Antibiotic 67-121C methyl ester hydrochloride having the following characteristics:

m.p.= 170°-180° (decomposition), λmax. (in tetrahydrofuran, water mixture, 4:1 parts by volume) 408 mμ (E_(1cm) ^(1%) = 750), 384 mμ (E_(1cm) ^(1%) = 970), 363 (E_(1cm) ^(1%) = 580).

The % chlorine found was 2.20% weight/weight.

By replacing the hydrochloric acid in the foregoing example with an equivalent quantity of other pharmaceutically acceptable acids, preferably mineral acids, other acid addition salts may be prepared. Exemplary of such acids are sulfuric, phosphoric, nitric, hydrobromic, acetic propionic, isobutyric, maleic, oxalic, phenylacetic, methanesulfonic or the like.

The following Examples exemplify some of the dosage forms in which the antibiotic products of this invention and their derivatives may be employed:

                  EXAMPLE 9                                                        ______________________________________                                         Tablets                                                                        Ingredient     5 mg. * Tab.                                                                              50 mg. * Tab.                                        ______________________________________                                         Antibiotic 67- 121                                                             complex        5.00 mg.   50.00 mg.                                            Lactose, U.S.P.                                                                               164.25 mg. 114.25 mg.                                           Corn starch    50.00 mg.  50.00 mg.                                            Polyvinylpyrrolidone                                                                          12.50 mg.  12.50 mg.                                            Magnesium stearate                                                                            1.25 mg.   1.25 mg.                                             Water q.s. (evaporates)                                                        ______________________________________                                    

PROCEDURE

Prepare a slurry consisting of the Antibiotic 67-121 complex, lactose, water and polyvinylpyrrolidone. Spray dry the slurry. Add the corn starch and magnesium stearate. Mix and compress into tablets.

                  EXAMPLE 10                                                       ______________________________________                                         Capsule                                                                        ______________________________________                                         Antibiotic 67-121C       5.00     mg.                                          Lactose U.S.P.           180.75   mg.                                          Magnesium stearate       1.25     mg.                                          Corn starch              46.00    mg.                                          Sodium lauryl sulfate    12.00    mg.                                                                   245.00   mg.                                          ______________________________________                                    

                  EXAMPLE 11                                                       ______________________________________                                         Oral Suspension (to give a dose of 25 mg/5 ml.)                                Antibiotic 67-121C tartrate                                                                           5.00      g.                                            Magnesium Aluminum Silicate                                                                           9.5       g.                                            SOdium Carboxymethylcellulose, U.S.P.                                                                 2.5       g.                                            Sodium Citrate, U.S.P. 25.0      g.                                            Flavor                 q.s.                                                    Color                  q.s.                                                    Methylparaben, U.S.P.  0.9       g.                                            Propylparaben, U.S.P.  0.2       g.                                            Polysorbate 80, U.S.P. 1.0       g.                                            Sorbitol Solution, U.S.P.                                                                             500.0     g.                                            Water     q.s.             1000.0    g.                                        ______________________________________                                    

PROCEDURE

1. Heat 200 ml. of water to boiling, and dissolve in it one half of the parabens. Cool to about 70° C., then mix in the Polysorbate 80. Sprinkle in the silicate, stirring until a uniform smooth suspension results.

2. Heat an additional 200 ml. of water to boiling, and dissolve in it the remainder of the parabens. Disperse the carboxymethylcellulose in this until a smooth gel results. Mix in the Sorbitol Solution. Then introduce and dissolve the sodium citrate.

3. Add 2 to 1 slowly, with constant stirring. Cool the mixture to 25° C. Add the antibiotic 67-121C, flavor and color, mixing thoroughly. Add sufficient quantity of water to make the total volume 1000 ml.

EXAMPLE 12 Topical Ointment

                  Example 12                                                       ______________________________________                                         Topical Ointment                                                               ______________________________________                                         Antibiotic 67-121C methyl ester                                                                           10      g.                                          Petrolatum                 990     g.                                                                     1000    g.                                          ______________________________________                                    

PROCEDURE

1. Melt the petrolatum.

2. Slurry the antibiotic 67-121C with about 10% of the petrolatum and pass through a colloid mill.

3. Mix the milled slurry with the remainder of the molten petrolatum. Allow to cool. 

We claim:
 1. A process for preparing Antibiotic 67-121, which comprises cultivating Antibiotic 67-121 producing species of Actinoplanes caeruleus having the indentifying characteristics of NRRL 5325 in an aqueous nutrient medium having assimilable sources of carbon and nitrogen under aerobic conditions until a composition of matter having substantial antifungal activity is produced and extracting Antibiotic 67-121 complex therefrom.
 2. A process according to claim 1 wherein the cultivation is effected at a temperature greater than 25° C. and less than 45° C. and including chromatographic means for separating said Antibiotic 67-121 complex into Antibiotic 67-121A, Antibiotic 67-121B, Antibiotic 67-121C and Antibiotic 67-121D.
 3. A polyene antibiotic complex designated Antibiotic 67-121 complex and having in free form substantially the following characteristics: C= 56.14, H= 7.53 and N= 1.79, M.P.° C.= 175, Rf= 0.43, ultra-violet absorption maxima in methanol at 342, 363, 382 and 403 mμ with corresponding E_(1cm) ^(1%) values of 340, 475, 667 and 605, infra red absorption maxima in mineral oil at 3350, 1725 and 1665 cm⁻ ¹ and nuclear magnetic resonance spectra in hexa-deutero-dimethylsulfoxide with maxima at 2.75, 6.56 and 7.64 ppm, said complex being in free form.
 4. A compound of claim 3, Antibiotic 67-121A, a yellow solid composition of matter, said compound having:(a) a melting point of 185°-190° C. dec; (b) an elemental analysis as follows: C= 59.40, H= 7.90 and N= 2.60%; (c) a molecular weight of 1218 by osmometry; (d) a specific rotation of +161.6° as measured by the D line of sodium at 26° C. and at 0.3% concentration in dimethylformamide; (e) ultraviolet maxima when measured in tetrahydrofuran H₂ O (4:1) at 408, 384, and 363 mμ with E_(1cm) ^(1%) = 950, 1100 and 680, respectively; (f) an infrared spectrum as a mineral oil (nujol) mull with characteristic absorption at 3350, 1725 and 1665 cm⁻ ¹ ; (g) a nuclear magnetic resonance spectrum with characteristic peaks at (d₆ -DMSO) 2.73 (3H, s, aromatic N-CH₃), 6.55 (2H, d, J= 8.5H_(z), aromatic H), and 7.64 PPM (2H, d, J= 8.5 H_(z), aromatic H); and (h) a pka of 6.0 (CO^(-O)), 9.0 (NH₃ ⁺) when measured in 66% aqueous dimethylformamide.
 5. A compound of claim 5, Antibiotic 67-121B, yellow solid composition of matter, said compound having:(a) a melting point of 175°-182° C dec; (b) The following elemental analysis: C= 59.70, H= 7.65, and N= 2.17% (c) a molecular weight of 1218 by osmometry; (d) a specific rotation of +199.7° as measured by the D line of sodium at 26° C. and at 0.3% concentration in dimethylformamide; (e) ultraviolet maxima when measured in tetrahydrofuran: H₂ O (4:1) at 408, 383 and 362 mμ with E_(1cm) ^(1%) = 620, 750 and 490, respectively; (f) an infrared spectrum as a mineral oil (nujol) mull with characteristic absorption at 3350, 1725 and 1666 cm⁻ ¹ ; (g) a nuclear magnetic resonance spectrum with characteristic peaks at δ (d₆ -DMSO) 6.58, (2H, d, J= 8.5Hz, aromatic H); and 7.60 PPM (2H, d, J= 3.5Hz, aromatic H); and (h) a pka of 6.3 (CO^(-O)), 9.0 (NH₃ ⁺) when measured in 66% aqueous dimethylformamide.
 6. A compound of claim 3, Antibiotic 67-121C, a yellow solid composition of matter, said compound having:(a) a melting point of 175°-180° C. dec; (b) the following elemental analysis: C= 57.06, H= 7.30, N= 2.07%; (c) a molecular weight of 1266 by osmometry; (d) a specific rotation of +143.8° as measured by the D line of sodium at 26° C. and at 0.3% in dimethylformamide; (e) ultraviolet maxima when measured in tetrahydrofuran H₂ O (4:1) at 408, 383 and 362 mμ with E_(1cm) ^(1%) = 800, 910 and 600, respectively; (f) an infrared spectrum as a mineral oil (nujol) mull with characteristic absorption at 3350, 1725 and 1665 cm⁻ ¹ ; (g) a nuclear magnetic resonance spectrum with characteristic peaks at (d₆ -DMSO) 2.76 (3H, s, aromatic N-CH₃), 6.57 (2H, d, J= 8.5H_(z), aromatic H) and 7.65 PPM (2H, d, J= 8.5H_(z), aromatic H); and (h) a pka of 6.0 (CO^(-O)), 9.0 (NH₃ ⁺) when measured in 66% aqueous dimethylformamide. 